Hyphenated Techniques Forum

The use of mass spectrometry detection with separation techniques such as liquid and gas chromatography has provided analysts with a much higher degree of specificity than that obtained using a single technique or instrument. Participants in this discussion of hyphenated techniques in chromatography are Nick Bukowski of ALMSCO; Monty Benefiel, Ken Miller, and Ed McCurdy of Agilent Technologies; Jim Edwards, Shona McSheehy, and Sergio Guazzotti of Thermo Fisher Scientific; and Ann Hoffman of Torion Technologies.

What new developments in LC–MS and GC–MS do you expect to see at Pittcon this year?

Bukowski:As with recent years, I'd expect to see further advances in MS technology directed towards the determination of trace levels of individual target compounds with a high degree of certainty. However, with the ever-expanding list of target compounds there is a limit to what MS alone can achieve. Therefore I would anticipate a rediscovery of the role of chromatography, enabled in part by developments in TOFMS,not in the form of higher resolving power, but rather as part of a multidimensional/comprehensive GC-MS solution, capable of screening the broadest suite of target compounds to trace levels in a single analysis. Such a powerful engine would also serve to monitor for the unexpected, as well as target compounds, and so associated with suchdevelopments, I would expect to see advances in software products to extract the relevant information in a " fit for use" form appropriate for the routine lab.

Benefiel:In the field of GC–MS, I expect to see continued advances in chromatographic and spectrometric selectivity. Identification and quantification of target compounds is enormously difficult if they are not adequately separated from closely related chemical inferences. This is why new developments in capillary flow devices, MS-MS analyzers, and deconvolution software are increasingly vital in the GC–MS analysis of real world samples.

Miller:We expect to see continuation of the general trend toward increasing sensitivity and increasing selectivity in MS analyses and the additional trend toward creation of “solutions” to address particular target markets. Sensitivity gains may come from improved inlet technology or improvements to MS design and hardware. Selectivity gains may come improved separation technology, sample prep, or improved MS data quality — for example, mass accuracy, resolution, acquisition rate, and so forth. “Solutions” arise mainly from kits, methods, and software advancements to facilitate very specific applications.

Edwards:A focus on robust, routine, systems at lower price points with solid, reliable performance; perhaps a focus on turn-key solutions which combine hardware, software, and chemistries/consumables predefined for certain applications. GC–MS systems, in particular, are tasked with higher and higher productivity requirements and systems that are specifically designed for maximum robustness and minimum downtime (for the rare instances of periodic maintenance) should be where the industry is moving to support their customers. Just as importantly, the software designed to utilize these systems must be focused on the requirements that are specific to a given area. Even though a GC–MS system may be used in an environmental or a toxicology laboratory, the software that is utilized by the operator for that work must “speak” to the requirements of those application areas in terms of nomenclature, workflow, and reporting options.

McSheehy:The use of HPLC–ICP-MS and GC–ICP-MS for elemental speciation is steadily growing as the analytical community recognizes that speciation analysis can provide a more accurate understanding of the sample under investigation. Whether it is in the environment, life sciences, or process control in industry, demand for speciation is on the rise. Elemental speciation has, for the last decade, been dominated by the environmental market and more recently assessing occupational exposure and risk from metals in foods through speciation in workplace, food, and clinical samples. Methodologies, including sample preparation protocols for speciation, are constantly being developed and improved and we see an increasing adoption of speciation techniques in contract laboratories where routine, robust, and reliable methods are essential.

Guazzotti:LC–MS is a field where technological advances will continue to improve instrumental performance. In terms of LC, I am sure Pittcon 2010 will see the introduction of developments that keep pushing the efficiency of chromatographic separations as well as the ability to carry out shorter runs that maintain desired chromatographic resolution and provide alternative ways to increase productivity. Some of the key developments to be introduced will certainly be related to pump technology, detector technology and column technology. For example, one of the obstacles when working with UHPLC has been the limitation of traditional UHPLC pumps to operate only in binary mode. Recent technological advances have permitted the development of UHPLC pumps with quaternary capabilities that provide performances that match or surpass those of traditional binary UHPLC pumps. This results in much higher flexibility in method development. I am sure we will continue to see developments in this direction as well as in improving pumping technology to be able to provide real flexible systems that are able to operate both in HPLC and UHPLC mode without sacrificing performance. With the further improvement of pumping technology we will most likely see a clear trend in the industry to provide systems that can operate at higher pressures than those that have been commonly used in UHPLC. As a consequence technical developments in other areas such as autosampler, detection, and column technologies will take full advantage of a wider dynamic range of operation of systems that can sustain much higher pressures. One improvement in LC instrumentation that we may see over the next few years is the development of smart systems that can monitor changes in system performance in real-time based on actual physicochemical measurements and the ability of these systems to adjust their behavior accordingly. In the case of LC–MS we will certainly keep seeing developments in the design of mass spectrometers, in particular of hybrid instruments that can provide high resolution and high sensitivity. The combination of UHPLC systems with expanded capabilities with this type of MS technology will certainly further the application of UHPLC–MS in high-throughput applications, such as those in drug development and discovery.

Hoffman:Instrumental developments for hyphenated systems have tended to be incremental — improved scan speeds, improved resolution, improved sensitivity, new ion sources, new inlet systems, and at the same time have become smaller, more economical, and easier to use. There is also significant movement toward high performance portable systems, especially GC–MS. Benchtop MS systems are now often smaller than the chromatographs with which they are interfaced, so smaller GC systems, perhaps with resistive column heating capabilities, will become more widespread. Comprehensive GCxGC–MS and LCxLC–MS systems are gaining momentum for analysis of complex mixtures as data handling software becomes powerful enough to handle the tremendous amount of data produced. Manufacturers are packaging solutions to topical analytical problems not only configured with recommended hardware with suitable performance specifications for the target application but also configured with complete workflows that include sample preparation protocols and software packages for data processing, interpretation, and reports generation.

Which industries currently benefit the most from the use of hyphenated techniques such as LC–MS and GC–MS?

Bukowski:These hyphenated techniques' reason for being is to address applications involving analysis of complex mixtures. No more important than in the determination and measurement of compounds hazardous to human health. So, hyphenated techniques are quintessential tools for those industries andagencies responsible for monitoring food safety, the environment - be it air, water, or soil - and consumer "home and personal care" products.

Benefiel and Miller:In general, any industry dealing with complex GC samples today will benefit most from the new advances in chromatographic and spectrometric selectivity discussed above. GC–MS is widely used in the environmental, food safety, and toxicological fields where low-level detection in complex matrices is critical.

Edwards:Any industry that has a need for the increased specificity that MS can provide over conventional GC detectors. These market areas include environmental and food safety, forensics, clinical, toxicology, and research. For example, environmental analysis can be done using regulated USEPA methods and both GC standalone and GC–MS single-quadrupole systems have a strong place in these areas. GC–MS is the technique of choice for certain methods that have a higher degree of specificity necessary to support the intended use of the data. Newer types of environmental and food safety analyses, which may task the system for performance in heavier matrices, larger numbers of target compounds, lower ultimate detection and with requirements for precise, accurate results, may look to GC–MS triple-quadrupole systems for their enhanced specificity capabilities. The same can be said of forensics, toxicology, and clinical testing — certain tests are ably handled by GC standalone systems (for example, blood alcohol testing), while other tests are more the domain of GC–MS single-quadrupole instruments (for example, pre-employment urine drug testing) or GC–MS triple-quadrupole and magnetic sector systems (for example, sports doping).

McSheehy:The drug and pharmaceutical industries are now appreciating that ICP-MS can sometimes provide a more sensitive detector than organic MS for some compounds. Although it’s unlikely we’ll see much published data on what they are actually looking at, we are seeing the increase in demand in this sector. Another industry that is using speciation to control processes and ultimately benefit from the information obtained by this technique is the petrochemical industry.

Guazzotti:Certainly one industry that has benefited from the use of LC–MS has been the pharmaceutical industry, where it is estimated that by the end of a trial phase as many as 90,000 LC–MS runs per compound can be performed. The introduction of high-speed LC provided the ability to significantly reduce analysis cycle times for LC–MS and to reduce the associated cost per sample. At the same time, while chromatographic runs became much shorter this resulted in a more efficient use of MS. In many cases, high-speed LC techniques, such as UHPLC, allowed for better chromatographic resolution to be obtained with the consequence of a significant improvement in the separation of analytes of interest from matrix compounds that can influence ionization in the MS system and affect sensitivity and reproducibility. Certainly in an industry where the cost and time associated with developing novel pharmaceutical compounds have significantly increased over the years, the implementation of techniques such as UHPLC–MS that can help reduce analysis time and provide advantages in many high-throughput applications has been very well received. The pharmaceutical industry continues to strive to reduce overall costs while improving standards of quality and reducing development time and therefore will continue to embrace technologies that can ultimately help improve the overall productivity of the process.

Hoffman:All industries involving analysis of complex mixtures or trace analysis in complex matrices greatly benefit from multidimensional techniques. The pharmaceutical industry is likely at the top of this list. The coupling of MS as the detector is potentially more sensitive, more specific, and more versatile than conventional detectors (UV, FID, and so forth). LC–MS-MS is the standard technique for quantifying trace analytes in biological samples for accurate pharmacokinetic profiling at the low pg/mL range. Also proteomics practitioners could not have made the same advances over the past two decades without the power of LC–MS. Other areas of application include environmental regulatory agencies for analysis of multitargeted environmental pollutants in a variety of matrices and characterization of complex biological samples in the food safety and natural products industries where high resolution separations and the specificity of mass spectrometry are required.

Has the increased use and availability of UHPLC had an impact on the technique of LC–MS?

Miller:Yes. UHPLC creates the ability to do fast, high-resolution separation of analytes before MS detection. A major benefit has been the ability to dramatically improve the throughput of LC–MS analyses by reducing the time required for the LC separation. A further benefit has been the ability to increase peak capacity and improve separations, leading to the detection and identification of more compounds in complex samples and to reduction of both ion suppression and matrix interference. UHPLC peaks are often very narrow compared to conventional LC peaks — maybe only 1-s wide. For accurate quantitation or comprehensive qualitative analyses it is essential that the MS system be able to sample each peak about 10 times and that there be no loss in MS performance. Most triple-quadrupole and TOF-based MS systems have adapted to this need, but it is still not possible with orbitraps.

Edwards:Yes, both in terms of MS design and in the application of the hyphenated technique. The narrower peak widths provided by UHPLC have driven the data acquisition speed requirements of mass spectrometers intended to be used for UHPLC–MS, while the improved chromatographic performance of UHPLC has made the systems both more productive (that is, accomplishing more analyses per unit time) and increased performance (that is, narrower, taller peaks allowing for improved signal-to-noise at lower concentrations).

Guazzotti:The increased use and availability of UHPLC has influenced the overall application and development of LC–MS. One example is the development and application of high-resolution MS in combination with UHPLC. The implementation of UHPLC has allowed significant reductions in overall analysis time while increasing chromatography resolution and sensitivity and reducing overall solvent consumption. An intrinsic effect of the use of UHPLC–MS is the associated increase in MS detection sensitivity per unit of volume injected due to the use of columns with smaller diameters, since the enhancement in MS signal is proportional to the ratio of the square of the diameters of the columns used for the chromatographic separation. Another consequence of the fast separations achieved with UHPLC is the need for optimizing data acquisition in the MS system. For example, if we consider that for quantitation we might want to have a certain number of data points across a peak (for example, 15–20), then the scan range selected in the MS system must be considered based on the overall chromatographic peak width and the scan speed. The scan range and scan speed certainly become very important parameters to be considered when carrying out UHPLC–MS, especially if we take into consideration that sensitivity and mass resolution can be sacrificed when data rates are increased. Developments in this area have been influenced by the increased utilization of UHPLC.

Hoffman:UHPLC introduction has had an impact on LC–MS with increased chromatographic efficiency producing sharper, narrower peaks with often greater sensitivity. The two primary target applications of UHPLC are analysis of complex mixtures and rapid high-volume analysis of less complex mixtures. UHPLC–MS is particularly useful for biomolecule analysis (such as peptides and proteins) in the high efficiency mode, and for pharmaceutical analysis in the high-speed (high-throughput) mode.

UHPLC utilizes small particles (